The feasibility of allogeneic bone marrow transplantation was demonstrated in children with severe osteogenesis imperfecta (Horwitz et al. 1999. Nat Med 5(3): 309-13). In that study, functional marrow-derived mesenchymal cells engrafted and contributed to the formation of new dense bone, indicating that the transplanted cells differentiated to bone-producing osteoblasts. Autologous bone marrow transplantation was also reported in a patient suffering from osteonecrosis of the humeral head (Hernigou et al. 1997. J Bone Joint Surg Am 79:1726-1730). Therefore, transplantation of stem cells capable of undergoing osteogenic differentiation or of cells that are committed towards osteogenic differentiation may be a promising avenue for the treatment of bone-related diseases, in particular when the treatment requires production of new bone.
Hence, there exists a great need for efficient techniques which provide sufficient quantities of cells, in particular autologous cells, suitable for transplantation as a remedy for bone-related disorders.
While undifferentiated bone marrow stem cells may be transplanted, these cells are not yet committed to an osteogenic lineage and therefore a considerable proportion of the transplanted stem cells may not eventually contribute to formation of bone tissue. In addition, it has been demonstrated (Banfi et al. 2000. Exp Hematol 28: 707-15) that in vitro culturing of bone marrow stem cells decreases their proliferation potential as well as their capability to undergo differentiation when treated with growth factors like FGF-2. For example, in that study, bone forming efficiency of in vitro cultured bone marrow stem cells was decreased 36 times already at first passage, compared to freshly isolated bone marrow. Hence, in vitro expansion of bone marrow stem cells may decrease their efficiency as a source of bone-forming osteoblasts upon transplantation.
Martin et al. (Endocrinology 138: 4456-4462, 1997) demonstrates that in vitro culturing of bone marrow stem cells in the presence of fibroblast growth factor type 2 (FGF-2), in combination with foetal calf serum (FCS) components, keeps the cells in an immature state (less alkaline phosphatase and fibroblast-like morphology), albeit the cells are competent to undergo osteogenic differentiation in vitro under specific osteogenic culture conditions. However, differentiation of such immature cells into bone-producing osteoblasts in vivo still depends on the provision of the appropriate signals upon transplantation. Hence, although such cells might be capable of osteogenic differentiation in vitro, a considerable proportion thereof may still not become osteoblasts in vivo. Also, Chaudhary et al. (Bone 34: 402-11, 2004) shows that human bone marrow stem cells treated with FGF-2 do not demonstrate any osteogenic phenotype (no alkaline phosphatase expression) and in fact have dystrophic morphology. Similarly, Kalajzic et al. (J Cell Biochem 88: 1168-76, 2003) demonstrates that FGF-2 inhibits osteogenic differentiation.
In addition, preparation of materials for use in human therapy should avoid the use of non-human animal components, such as serum components (e.g., FCS) in the culture media. However, as shown by Kuznetsov et al. (Transplantation 70: 1780-1787, 2000), the use of homologous or autologous human sera greatly diminishes the ability of human bone marrow stem cells to form colonies and expand in vitro, and to form bone in vivo. Hence, Kuznetsov et al. suggest that the use of FCS is prerequisite for efficient expansion of bone marrow stem cells and for their capacity to form bone.
Takagi et al. 2003 (Cytotechnology 43: 89-96) incubated human bone marrow aspirates in donor serum supplemented with FGF-2, under specific conditions. The cell population so-obtained by Takagi et al. 2003 only showed chondrogenic differentiation potential and was thus contemplated by the authors for use in the regeneration of cartilage.
Kobayashi et al. 2005 (J Bone Joint Surg Br 87: 1426-3) describes particular conditions for isolation and maintenance of human BMSC in autologous donor serum, concluding that these conditions may provide for sufficient ex vivo expansion of human BMSC, while preserving their multi-differentiation potential. FGF-2 is employed by Kobayashi et al. 2005 in secondary culture to promote further BMSC expansion without differentiation. These authors do not disclose conditions which would cause their cells to progress towards osteoprogenitors or osteoblast phenotype cells.
Lin et al. 2005 (Transplant Proc 37: 4504-5) reported prolonged expansion of multi-potential human BMSC in autologous donor serum. Addition of FGF-2 and EGF to the cells under certain conditions did not influence cell proliferation and did not cause progression of the cells towards osteogenic fate.
In order to provide for maximum bone formation, it would be desired to transplant cells which already show an osteoblastic phenotype, since such cells are essentially the only ones with a demonstrated bone-forming activity. However, in vitro differentiation of bone marrow stem cells into osteoblasts involves culturing in osteogenic medium (Jaiswal et al. 1997. J Cell Biochem 64: 295-312) and may lead to decreased proliferation of such cells in vitro. Moreover, the use of osteogenic medium involves addition of further components to the cells, which may increase the risk of contamination of the cell culture.
Hence, there exists a need in the art for a simple and reliable method to produce osteoprogenitors, osteoblasts or osteoblastic phenotype cells from human adult stem cells, in particular human bone marrow stem cells, in vitro while maintaining high expansion capacity of the cells, ensuring autologous conditions and minimising the number of components involved in culturing of the cells.
There also exists need in the art for osteoprogenitor or osteoblastic cells having specific useful characteristics, e.g., in the context of bone therapy, and for cell populations comprising such cells.